Method of producing lys-plasminogen

ABSTRACT

To produce lys-plasminogen having a specific activity of at least 17.5 caseinolytic units/mg protein and at least 50 mymoles/g protein nitrogen as well as an electrophoretic purity of at least 90%, plasminogen from plasma, a plasminogen-containing fraction or a tissue culture is adsorbed on immobilized lysine for the purpose of purification, is eluted and is recovered from the eluate by a protein precipitating agent. A solution of the thus purified plasminogen is adjusted to a plasmin activity ranging between 0.005 and 0.2 mymoles/ml min relative to chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, is maintained at a temperature of from +1° C. to +20° C. for a period of from 6 to 60 hours in order to provoke an enzymatic-proteolytic conversion of plasminogen into lys-plasminogen, whereupon the enzymatic action is interrupted and lys-plasminogen is isolated.

This application is a continuation of application Ser. No. 07/900,794,filed Jun. 22, 1992, now abandoned, which is a continuation ofapplication Ser. No. 07/378,277, filed Jul. 11, 1989, now abandoned.

The invention relates to a method of producing lys-plasminogen having aspecific activity of at least 17.5 caseinolytic units/mg protein and atleast 50 μmoles/g protein nitrogen as well as an electrophoretic purityof at least 90%, wherein plasminogen from plasma, aplasminogen-containing fraction or a tissue culture is adsorbed onimmobilized lysine for the purpose of purification, is eluted and isrecovered from the eluate by a protein precipitating agent.

Lys-plasminogen is a generic name used in the literature to denoteproteolytically modified forms of native plasminogen (glu-plasminogen)which are obtained from the same by cleaving a polypeptide from the NH₂terminal. As the N-terminal amino acids of the presently known speciesof lys-plasminogen, lysine, methionine and valine have been detected sofar. Values of between 90,000 and 94,000 for glu-plasminogen and valuesof about 80,000 for lys-plasminogen have been indicated in theliterature as the respective molecular weights.

With regard to the fibrinolytic behavior, lys-plasminogen differs fromglu-plasminogen primarily in two respects: lys-plasminogen possesses ahigher binding affinity to the fibrin networks of thrombi and can beactivated to plasmin far more rapidly (e.g., by urokinase). Bothproperties increase the efficiency of fibrinolysis and are the reasonsfor lys-plasminogen preferably being used in thrombosis therapy.

The starting material for the recovery of lys-plasminogen is the Cohnfraction III of plasma or plasma itself. The processes described in theliterature consist of a plurality of process steps, such as extraction,adsorption and precipitation steps. The cleavage of an N-terminalpolypeptide chain and conversion into lys-plasminogen either is effecteddirectly on isolated glu-plasminogen or takes place in the course of theisolation and purification steps on account of the proteolytic activitypresent.

P. Wallen and B. Wiman (Biochim. Biophys. Acta 221, 20-30 (1970))isolated plasminogen by extracting a washed Cohn III precipitate andprecipitating the extract with methanol and ammonium sulfate at pH 4.8.This product contains 0.5 to 1.9% spontaneous proteolytic activity dueto plasmin contaminations. After the addition of aprotinin, furtherpurification by means of gel filtration and adsorption/elution on ananion exchanger yields a plasminogen that predominantly exhibitsglutamine as the N-terminal amino acid and has a spontaneous proteolyticactivity of 0.1 to 0.3%. Processing without any addition of aprotiningives rise to a product mixture having a spontaneous proteolyticactivity of 4% and lysine, valine, but also methionine and glutamine asN-terminal amino acids.

E. E. Rickli and P. A. Cuendet (Biochim. Biophys. Acta 250, 447-451(1971)) immobilized lysine on polyacrylamide gel, using this gel toisolate plasminogen from plasma. After washing with a phosphate buffer,elution takes place with a buffer containing 0.5 moles/l 6-aminocaproicacid. By adding ammonium sulfate, plasminogen is precipitated from theeluate and the dissolved precipitate is dialyzed againstNaCl-Tris-lysine or phosphate-NaCl buffer. Only glutamine is detectableas N-terminal amino acid.

D. Collen et al. (Thromb. Res. 7, 515-529 (1975)) also describe thepreparation of plasminogen by affinity chromatography with lyeineagarose from plasma or Cohn III precipitate with and without theaddition of aprotinin. Thus, with aprotinin pure glu-plasminogen isrecovered from plasma, whereas without aprotinin the product containstraces of proteins with lys and val as N-terminal amino acids. Withoutany addition of aprotinin, a preparation with a spontaneous proteolyticactivity (induced by the plasmin present) of 0.1 to 1% was isolated fromCohn III precipitate. When adding 25,000 to 50,000 KIU aprotinin per kgCohn III precipitate and 5 KIU/ml to the washing and eluting solutions,the spontaneous proteolytic activity was below 0.1%, the separation ofglu- and lys-plasminogen was effected by ion exchange chromatography onDEAE Sephadex A-50.

H. Claeys et al. (Thromb. Res. 3, 515-523 (1973) and Biochim. Biophys.Acta, 342, 351 (1974)) extracted plasminogen from Cohn III precipitatewith a phosphate buffer (0.1 mole/l, pH 7.4) containing 10 KIUaprotinin/ml. After adsorption on lysine agarose and washing with aphosphate buffer, elution takes place with a buffer containing6-aminocaproic acid (0.2 moles/l) and aprotinin (20 KIU/ml). The thusisolated plasminogen had a spontaneous proteolytic activity of about0.4% and, in addition to glu and lys, contained traces of val and met asN-terminal amino acids. When processing plasma according to the sametechnique, only glu is detected as N-terminal amino acid, thespontaneous proteolytic activity amounting to below 0.1%. Furtherpurification is effected by gel filtration (Sephadex G-150) and ionexchange chromatography (DEAE-Sephadex A-50). When adding aprotinin (400KIU/ml) prior to further purification, an end product is isolated evenwith Cohn III precipitate used as starting material, which end productexhibits a spontaneous proteolytic activity of below 0.1% and glu asN-terminal amino acid. The conversion of glu-plasminogen tolys-plasminogen is achieved by the authors by incubation with plasmin(plasminogen/plasmin 200:1 to 20:3) at 37° C. and is stopped by theaddition of trichloroacetic acid or aprotinin.

All these methods have disadvantages, because either they are not suitedfor the large-scale isolation of lye-plasminogen comprising methodsteps, such as, e.g. gel filtration, or they must be performed at roomtemperature, thus involving the risk of bacterial contamination of thepharmaceutical preparation. Usually, these methods, moreover, give riseto preparations that contain plasmin and, thus, have a high portion ofspontaneous proteolytic activity. This is exactly what is undesired froma therapeutical point of view, because, upon application of thepreparations, it takes effect on proenzymes of the coagulation system,partially destroying the same and, thus, giving rise to undesired sidereact ions. Although this proteolytic activity may be inhibited, thiswill, in turn, result in the contamination of the formed inactivecomplexes of the preparations produced.

The invention aims at avoiding these disadvantages and has as its objectto produce lys-plasminogen having a specific activity of at least 17.5caseinolytic units per mg protein and at least 50 μmoles plasminogen pernitrogen as well as an electrophoretic purity of at least 90%, whereinplasminogen from plasma, a plasminogen-containing fraction or a tissueculture is adsorbed on immobilized lysine for the purpose ofpurification, is eluted and is recovered from the eluate by a proteinprecipitating agent.

According to the invention, this object is achieved in that a solutionof the thus purified plasminogen is adjusted to a plasmin activityranging between 0.005 and 0.2 μmoles/ml min, preferably 0.01 and 0.1μmoles/ml min, relative to the chromogenic substrateH-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, ismaintained at a temperature of from +1° C. to +20° C., preferably offrom +4° C. to +12° C., for a period of from 6 to 60 hours, preferably15 to 50 hours, in order to provoke an enzymatic-proteolytic conversionof plasminogen into lys-plasminogen, whereupon the enzymatic action isinterrupted and lys-plasminogen is isolated.

To adjust the plasmin activity in accordance with the invention,plasmatic enzymes of the group consisting of serine proteases may beused. They comprise plasmin and plasmin-like enzymes. A plasmin activityof 0.01 to 0.1 μmoles/ml min corresponds to a plasminogen/plasmin ratioof from 30,000:1 to 3,000:1. Hence it is apparent that the proteolyticconversion of glu-plasmingen into lys-plasminogen is effected withsurprisingly small amounts of enzyme.

Preferably, the action of plasmin on plasminogen is interrupted by theaddition of inhibitors.

By using extremely slight amounts of plasmatic enzyme, the methodaccording to the invention also does with slight amounts of inhibitorsto interrupt the enzymatic action. By the addition of, e.g., 10 to 200KIU/ml aprotinin or 0.01 to 1.0 U/ml Cl-esterase inhibitor, the activitymay be lowered to less than or equal to 0.03 μmoles/ml relative toSubstrate S 2251. This value is sufficient to ensure the stability ofthe product during subsequent processing steps, such as filtration,freeze-drying and, if desired, heat treatment for the inactivation ofpossibly present bacteria or viruses.

To restrict the action of plasmin, also other inhibitors, such asalpha2-macroglobuline or alpha2-antiplasmin, may be used.

A favorable embodiment of the method according to the invention consistsin that the action of plasmin on plasminogen is effected while carryingout dialysis.

A particularly favorable variant of the method according to theinvention for producing heat-stable lys-plasminogen from blood plasma orblood plasma products, which has a specific activity of at least 17.5caseinolytic units per mg protein, at least 50 nmoles plasminogen per mgnitrogen as well as an electrophoretic purity of at least 90%, ischaracterized by the combination of the following measures:

a) extracting a Cohn fraction III (precipitate) by means of a phosphatebuffer in the presence of 0.1 to 100 KIU aprotinin per ml buffersolution in order to obtain a crude plasminogen fraction,

b) treating the crude plasminogen fraction with ethanol in order toprecipitate the main portion of non-plasminogen proteins from theextract and eliminate the same,

c) isolating plasminogen from the ethanolic solution by adsorption onimmobilized lysine and subsequent elution,

d) precipitating the thus purified plasminogen by means of ammoniumsulfate or polyethylene glycol (PEG) and centrifuging the same,

e) dissolving the precipitate and dialyzing the solution in the presenceof plasmin, the solution having a plasmin activity of from 0.01 to 0.1μmoles/ml min relative to chromogenic substrate S 2251, then

f) interrupting the enzymatic action by adding 10 to 200 KIU aprotinin,and

g) lyophilizing the solution and, if desired, heat-treating thelyophilisate.

This embodiment, thus, departs from a Cohn fraction III (precipitate),an extract being initially produced from this fraction with a phosphatebuffer. This extract contains large amounts of inert accompanyingproteins, such as, e.g., immunoglobulins and lipoproteins, the latter,in particular, rendering difficult the purification by means of affinitychromatography on immobilized lysine, due to poor filtration propertiesand unspecific interactions.

By precipitation with ethanol, the major portion of these disturbingaccompanying proteins can be eliminated.

On account of the presence of 0.1 to 100 KIU aprotinin/ml buffersolution, the stability of plasminogen is safeguarded during affinitychromatographic purification. As the matrix for affinity chromatography,a gel containing lysine as the ligand, preferably a lysine-polyacrylamide gel, is used. After adsorption, the remaining accompanyingproteins are removed by washing--preferably by means of a phosphatebuffer--and subsequently plasminogen is eluted. This affinitychromatographic purification may be carried out either on a column or ina batch process.

By treating the eluate with protein precipitating agents, such as, e.g.,ammonium sulfate or polyethylene glycol 4000, an intermediate product isprecipitated that corresponds to glu-plasminogen described in theliterature in terms of its properties and, in SDS PAGE, has a molecularweight of 92,000.

This intermediate product is capable of being converted into afibrinolytic proenzyme already with extremely slight amounts of plasmin,which proenzyme exhibits the properties of lys-plasminogen described inthe literature and, in SDS-PAGE, has a molecular weight of 84,000.

By optionally adding plasmin or aprotinin, the proteolytic activity ofthe solution of purified plasminogen (glu-plasminogen) may be increasedor lowered such that the above-defined range of preferably 0.01 to 0.1μmoles/ml min will be very easily adjustable in this manner. Theenzymatic action within the solution is interrupted after the conversionwith aprotinin, the solution is lyophilized and, if desired, thelyophilisate is heat-treated.

The procedures necessary to carry out the method according to theinvention will be explained in more detail in the following.

Determination of the spontaneous proteolytic activity with chromogenicsubstrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (S2251, Kabi):

The principle consists in that plasmin and other proteolytic enzymescleave from the chromogenic substrate p-nitroaniline, whose liberationis measurable spectrophotometrically as the increase in extinction at405 nm.

At first, the samples are pre-diluted at ratios of 1:2, 1:5, 1:10 and1:20, to which end an aqueous solvent having pH=7.2 and containing 10mmoles/l lysine, 0.5% by weight PEG 6000 and 50% by weight glycerol isused.

The chromogenic substrate is dissolved to a concentration of 5 mmoles/lin distilled water. An aqueous solution of pH=7.2 containing 50 mmoles/lTris and 180 mmoles/l NaCl serves as the test buffer.

For assaying, 0.25 ml sample dilution is mixed with 0.55 ml test buffer,is incubated at 37° C., and, upon the addition of 0.05 ml substratesolution, the increase in the extinction at 405 nm is observed at atemperature of 37° C. The calculation of the activity is based on theformula

    E/min.0.321.dilution factor=μmoles/ml min

Determination of the specific activity of plasminogen:

a) Caseinolytic units (CU)/mg protein

By reaction with streptokinase, plasminogen is activated to plasmin,which cleaves casein fragments that are soluble in trichloroacetic acidand are detectable by spectrophotometric measurement of the extinctionat 280 nm in the supernatant.

The pre-dilution of the samples to an activity of 0.5 to 2.5 CU/ml iseffected with an aqueous solution of pH=9.0, containing 50 mmoles/lTris, 20 mmoles/l lysine, 0.1 moles/l NaCl and 1 mmoles/l EDTA.

The substrate is produced by dissolving casein according to Hammarsten(4%) in a phosphate buffer (67 mmoles/l, pH 7.4).

For activation, a solution of streptokinase (2500 IU/ml) in phosphatebuffer as above is used .

To effect assaying 2.0 ml casein solution, 1.4 ml phosphate buffer (67mmoles/l, pH 7.4), 0.4 ml sample dilution and 0.2 ml streptokinasesolution are mixed in an ice-bath, buffer being pipetted instead of thesample dilution for the blank. After heating to 37° C. for 30 minutes,the samples are again placed into the ice-bath and the non-cleavedcasein is precipitated by the addition of 6 ml 15% trichloroacetic acid.After standing for at least 30 minutes at room temperature, filtrationover folded filters takes place and the extinction in the supernatant ismeasured at 280 nm relative to the blank.

The calculation of the caseinolyic activity is effected according to theformula

    E.sub.280.16.3dil.=CU/ml.

Double assays are each made of at least two different dilutions beingwithin the measuring range.

The conversion into the specific activity (CU/mg protein) is effected bydividing the caseinolytic activity by the protein value (Kjeldahl, inmg/ml).

b) Micromole plasminogen/g protein nitrogen

This assay method is known as "active site cross titration". At first,plasminogen is activated by complex formation with streptokinase. Thiscomplex reacts with p-nitrophenol-p'-guanidino benzoate under liberationof p-nitrophenol, which is determined spectrophotometrically bymeasuring the extinction at 400 nm.

The following reagents are used:

    ______________________________________                                        Test buffer (pH = 7.7):                                                                     100 mmoles/1 Na.sub.2 HPO.sub.4                                                1 mmole/1 6-aminocaproic acid                                  Streptokinase buffer:                                                                        30 mmoles/1 Na.sub.2 HPO.sub.4                                               150 mmoles/1 Na glutamate                                       Titration solution:                                                                          2.5 mmoles/1 p-nitrophenyl-p'-                                               guanidino benzoate in dry DMSO                                                (dried over molecular sieve 0.4 nm)                             Standard solution:                                                                          1.0 mg p-nitrophenol per 1 ml in dry                                          DMSO. Prior to utilization, 5 ml are                                          diluted with dry DMSO to 100 ml.                                ______________________________________                                    

For assaying, ml test buffer, 50 μl streptokinase solution (about 200nmoles/ml) and "V" μl sample are incubated at 25° C. for 20 to 30 min,the extinction being measured at 400 nm for 2.5 minutes. After theaddition of 20 μl titration solution, it is mixed, the extinction againbeing observed at 400 nm. The increase in extinction measured after theaddition of the titration solution, which is to range between 0.08 and0.12, is entered into the calculation as "At".

Blank run: Instead of streptokinase, streptokinase buffer and, insteadof the sample, an equal volume of a buffer having the same saltcomposition as the sample are added, otherwise the procedure is the sameas in the test run. The increase in extinction is entered in thecalculation as "Ab".

Standard run: Procedure as in the blank run, yet instead of thetitration solution, a standard solution is added. The increase inextinction is entered in the calculation as "As".

The calculation of the plasminogen activity is effected according to theformula ##EQU1##

Dividing this activity by the content of the solution of proteinnitrogen (Kjeldahl, in g/l) yields the specific activity of plasminogenin μmole/g protein nitrogen.

Determination of the molecular weight (SDS polyacrylamide gelelectrophoresis, SDS PAGE):

This method consists in that proteins are uniformly charged by theaddition of SDS and migrate according to their molecular sizes inpolyacrylamide gel when applying electric potential. By comparing themigration distance with that of calibration proteins of known molecularweights, the molecular weight of the samples to be assayed can bedetermined.

To carry out the method, 47.5 g acrylamide and 2.5 gN,N-methylene-bis-acrylamide are dissolved in an aqueous buffer solutionof pH 7.1 containing 6 moles/l urea, 0.1 moles/l Tris and 0.1% SDS(sodium dodecyl sulfate).

57 ml of this solution, 3 ml of a 0.5% aqueous ammonium peroxo disulfatesolution and 90 myl N,N,N',N'-tetramethyl ethylenediamine are rapidlymixed and filled into a gel casting stand. After polymerization, the gelis used in an electrophoresis apparatus. The samples are diluted to aprotein concentration of 2.5 mg/ml with a solution containing 9 moles/lurea and 4.5% SDS. After further dilution (1+1) with glycerol bromphenolblue, 6 μl sample are each applied. A buffer of pH=7.1 containing 0,1mole/l Tris, 0.1% SDS and 0.001% sodium azide is used forelectrophoresis. The electrophoresis is performed for 10 min at 50 V andsubsequently for 3 h at 100 V. Then the gel is stained with a solutionof 2 g Coomassie blue in 455 ml methanol, 455 ml water and 90 ml glacialacetic acid for one hour at room temperature. Destaining is effected bya mixture of 2.5 l methanol, 1.0 l glacial acetic acid and 6.5 l water.

To determine the molecular weight, the migration distances are measured,the latter being proportional to the logarithm of the molecular weight.From a parallelly analyzed calibrator, a calibration line is, thus,established, from which the molecular weight of the samples to beinvestigated may be read.

If several protein bands are to be recognized in the samples to beinvestigated, their distribution will be determined by densitometricevaluation of the extinction at 601 nm.

Preparation of lysine polyacrylamide gel:

100 g Biogel P-300 (150-300 μm, polyacrylamide gel of Bio-Rad) areswelled in 2.6 l water for 2 h at 47° C. and are converted into thehydrazide by reaction with 1.2 l hydrazine hydrate during 20 h at 47° C.It is washed with distilled water until a pH of below 8.5 is reached(filtration over Buchner funnels). The washed gel is suspended in 6 l0.3N HCl and the pH is corrected to 1.1. After cooling to 0° C., a coldsolution of 56 g sodium nitrite in 200 ml water is added and stirred for3 min. After the addition of a solution of 730 g lysine in 2 l water, itis stirred for at least 3 h at a temperature of not greater than +4° C.and at a pH of 9.5. Subsequently, stirring is continued for further 16 hat +4° C. After washing three times with 20 l water each and filtrationover Buchner funnels, it is stirred with 15 l of a solution containing107 g/l ammonium chloride (pH=8.8) for 16 to 20 h at +4° C. Then the gelis washed with water and afterwards with a phosphate buffer.

The method according to the invention will be further explained by wayof the following examples.

EXAMPLE 1

1 kg Cohn III precipitate was suspended at 0° C. in 10 l of a phosphatebuffer pH 7.4 to which 10 KIU/ml aprotinin had previously been added.Under cooling to -2° C., ethanol was added until a final concentrationof 10%. After 15 hours of stirring at a temperature of -2° C., it wascentrifuged and the supernatant was filtered over a depth filter basedon cellulose (AMF Cuno Zeta Plus 50 S). Subsequently, it was dilutedwith 5 l phosphate buffer and 500 g lysine polyacrylamide gel wereintroduced. After a stirring time of one hour at 0° C., the gel loadedwith plasminogen was separated by filtration over Buchner funnels andwashed with a phosphate buffer several times until no more protein wasdetectable in the filtrate. By stirring with a solution of6-aminocaproic acid (0.1 moles/l) in phosphate buffer, plasminogen waseluted and subsequently precipitated by the addition of 261 g ammoniumsulfate per kg eluate.

The precipitate obtained by centrifugation was dissolved in 32 ml of anisotonic phosphate/saline buffer. Since further processing of thepreparation according to the invention depends on the presence of aplasmatic enzyme, i.e., plasmin, having the activity indicated, theplasmin activity of the solution relative to chromogenic substrate S2251 was measured at first. It amounted to 0.045 μmoles/ml so that nocorrectional adjustment was necessary in that case. In SDS PAGE only oneband at a molecular weight of 92,000 (=glu-plasminogen) was detectableprior to carrying out the subsequently intended dialysis.

After dialysis against an isotonic phosphate/saline solution for 36 h ata temperature of 5.0° C., 50 KIU/ml aprotinin were added. The activityrelative to substrate S 2251 amounted 0.01 μmoles/ml min at that time.The test for caseinolytic activity revealed 342 CU/ml. With a proteincontent of 15.6 g/l (according to Kjeldahl), this corresponds to aspecific activity of 21.9 CU/mg. The active site cross titrationrevealed an activity of 164 μmoles/l and, hence, a specific activity of65.7 μmoles/g protein nitrogen. During dialysis, the conversion of theprotein having a molecular weight of 92,000 (glu-plasminogen) into aprotein having a molecular weight of 84,000 (lys-plasminogen) wasdetecable in SDS PAGE. The portion of this product was 98%.

After the addition of 20 mmoles/l lysine, a pH of 7.0 was adjusted, thenfiltration and freeze-drying were carried out. The water content of thelyophilisate was brought to 7.8% by weight, and subsequently it washeated to 60° C. for 10 h to inactivate possibly present bacteria orviruses. After dissolution in distilled water, it was sterile-filtered,sterile-filled and again freeze-dried. In the tests for specificactivity and electrophoretic purity, the thus obtained final productshowed unaltered results compared to the product obtained immediatelyupon dialysis.

EXAMPLE 2

The extraction of Cohn III precipitate, ethanol precipitation andfiltration were carried out as in Example 1. After dilution withphosphate buffer, the solution was pumped through a chromatographiccolumn packed with 100 g lysine polyacrylamide gel. Washing and elutionwere performed with the solutions described in Example 1, but in thechromatographic column. Precipitation from the eluate with ammoniumsulfate and dissolution of the precipitate were effected as inExample 1. In SDS PAGE only one band at a molecular weight of 92,000 wasdetected. The activity relative to chromogenic substrate S 2251 amountedto 0.003 μmole/ml min. By adding plasmin, it was increased to 0.016μmole/ml min. After dialysis for 43 h at 7° C. and subsequent additionof 100 KIU/ml aprotinin, the portion of the band at a molecular weightof 84,000 (=lys-plasminogen) was found to be more than 98% in SDS PAGE.The specific activity amounted to 22.3 CU/mg protein and 60.5 μmoles/gnitrogen, respectively.

EXAMPLE 3

The extraction of Cohn III precipitate with phosphate buffer was carriedout after the addition of 2 KIU/ml aprotinin. Further processing untilprecipitation with ammonium sulfate and dissolution of the precipitatewere effected as in Example 2.

The activity of the solution relative to chromogenic substrate S 2251amounted to 0.014 μmole/ml min. After dialysis for 22 h at a temperatureof 11.7° C. and subsequent addition of 20 KIU/ml aprotinin, a specificactivity of 20.5 CU/mg protein and 56.3 μmole/g protein nitrogen,respectively, was found, the portion of lys-plasminogen in SDS PAGE was96%.

EXAMPLE 4

The isolation of plasminogen until the elution of lysine polyacrylamidegel was effected as described in Example 3. By the addition of 70 gpolyethylene glycol 4000 to 350 ml eluate, plasminogen was precipitated.The precipitate obtained by centrifugation was dissolved in 15 ml of aphosphate/NaCl buffer; in SDS PAGE only "glu-plasminogen" having amolecular weight of 92,000 was detectable. After the addition ofplasmin, the activity measured with substrate S 2251 amounted to 0.01μmole/ml min; dialysis took place for 42 hours at a temperature of 6.6°C., subsequently aprotinin (50 KIU/ml) was added.

The portion of lys-plasminogen in SDS PAGE was 95%; a specific activityof 23.4 CU/mg protein and of 59.0 μmole/g protein nitrogen,respectively, was determined.

What we claim is:
 1. A method of producing lys-plasminogen having a specific activity of at least 17.5 caseinolytic units/mg protein and at least 50 μmols/g protein nitrogen as well as an electrophoretic purity of at least 90% comprising the steps of:(i) adsorbing plasminogen from plasma, a plasminogen-containing fraction or a tissue culture on immobilized lysine for the purpose of purification; (ii) eluting said plasminogen; (iii) precipitating said plasminogen from the eluate; (iv) dissolving the precipitated plasminogen to obtain a solution containing plasminogen and trace amounts of plasmin activity; (v)(a) if the plasmin activity is between 0.005 and 0.2 μmol/ml min relative to chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, plasminogen solution is maintained at a temperature of from +1° C. to +20° C. for a period of from 6 to 60 hours in order to provoke an enzymatic-proteolytic conversion of said plasminogen into lys-plasminogen; (v)(b) if the plasmin activity is below 0.005 μmol/ml min relative to said chromogenic substrate, plasmin is added to obtain a plasmin activity between 0.005 and 0.2 μmol/ml min relative to said chromogenic substrate and plasminogen solution is maintained at a temperature of from +1° C. to 20° C. for a period of from 6 to 60 hours in order to provoke an enzymatic-proteolytic conversion of said plasminogen into lys-plasminogen; or (v)(c) if the plasmin activity is above 0.2/μmol/ml min relative to said chromogenic substrate, plasmid inhibitor is added to obtain a plasmin activity between 0.005 and 0.2 μmol/ml min relative to said chromogenic substrate and the plasminogen solution is maintained at a temperature of from +1° C. to 20° C. for a period of from 6 to 60 hours in order to provoke an enzymatic-proteolytic conversion of said plasminogen into lys-plasminogen; (vi) interrupting the enzymatic action with a plasmin inhibitor; and (vii) isolating said lys-plasminogen.
 2. The method of claim 1, wherein said plasmin activity ranges between 0.01 and 0.1 μmole/ml min, said temperature ranges from +4° C. to +12° C. and said period of time ranges from 15 to 50 hours.
 3. The method of claim 1, wherein said plasmin inhibitor is selected from the group consisting of aprotinin in an amount of from 10 to 200 KIU/ml, Cl-esterase inhibitor in an amount of from 0.01 to 1.0 U/ml, alpha2-macroglobulin and alpha2-antiplasmin.
 4. The method of claim 1, wherein said plasmin activity is allowed to act on said plasminogen while carrying out dialysis.
 5. The method of claim 1 wherein said plasmin activity ranges between 0.01 and 0.1 μmol/ml min. 